Polyketides represent a large family of diverse compounds synthesized from 2-carbon units through a series of condensations and subsequent modifications. Polyketides occur in many types of organisms, including fungi and mycelial bacteria, in particular, the actinomycetes. There are a wide variety of polyketide structures, and the class of polyketides encompasses numerous compounds with diverse activities. Erythromycin, FK-506, FK-520, megalomicin, narbomycin, oleandomycin, picromycin, rapamycin, spinocyn, and tylosin are examples of such compounds. Given the difficulty in producing polyketide compounds by traditional chemical methodology, and the typically low production of polyketides in wild-type cells, there has been considerable interest in finding improved or alternate means to produce polyketide compounds. See PCT publication Nos. WO 93/13663; WO 95/08548; WO 96/40968; 97/02358; and 98/27203; U.S. Pat. Nos. 4,874,748; 5,063,155; 5,098,837; 5,149,639; 5,672,491; 5,712,146; and 5,962,290; and Fu et al., 1994, Biochemistry 33: 9321-9326; McDaniel et al., 1993, Science 262: 1546-1550; and Rohr, 1995, Angew. Chem. Int. Ed. Engl. 34(8): 881-888, each of which is incorporated herein by reference.
Polyketides are synthesized in nature by polyketide synthase (PKS) enzymes. These enzymes, which are complexes of multiple large proteins, are similar to the synthases that catalyze condensation of 2-carbon units in the biosynthesis of fatty acids. PKS enzymes are encoded by PKS genes that usually consist of three or more open reading frames (ORFs). Two major types of PKS enzymes are known; these differ in their composition and mode of synthesis. These two major types of PKS enzymes are commonly referred to as Type I or “modular” and Type II “iterative” PKS enzymes. A third type of PKS found primarily in fungal cells has features of both the Type I and Type II enzymes and is referred to as a “fungal” PKS enzymes.
Modular PKSs are responsible for producing a large number of 12-, 14-, and 16-membered macrolide antibiotics including erythromycin, megalomicin, methymycin, narbomycin, oleandomycin, picromycin, and tylosin. Each ORF of a modular PKS can comprise one, two, or more “modules” of ketosynthase activity, each module of which consists of at least two (if a loading module) and more typically three (for the simplest extender module) or more enzymatic activities or “domains.” These large multifunctional enzymes (>300,000 kDa) catalyze the biosynthesis of polyketide macrolactones through multistep pathways involving decarboxylative condensations between acyl thioesters followed by cycles of varying β-carbon processing activities (see O'Hagan, D. The polyketide metabolites; E. Horwood: New York, 1991, incorporated herein by reference).
During the past half decade, the study of modular PKS function and specificity has been greatly facilitated by the plasmid-based Streptomyces coelicolor expression system developed with the 6-deoxyerythronolide B (6-dEB) synthase (DEBS) genes (see Kao et al., 1994, Science, 265: 509-512, McDaniel et al., 1993, Science 262: 1546-1557, and U.S. Pat. Nos. 5,672,491 and 5,712,146, each of which is incorporated herein by reference). The advantages to this plasmid-based genetic system for DEBS are that it overcomes the tedious and limited techniques for manipulating the natural DEBS host organism, Saccharopolyspora erythraea, allows more facile construction of recombinant PKSs, and reduces the complexity of PKS analysis by providing a “clean” host background. This system also expedited construction of the first combinatorial modular polyketide library in Streptomyces (see PCT publication Nos. WO 98/49315 and 00/024907, each of which is incorporated herein by reference).
The ability to control aspects of polyketide biosynthesis, such as monomer selection and degree of β-carbon processing, by genetic manipulation of PKSs has stimulated great interest in the combinatorial engineering of novel antibiotics (see Hutchinson, 1998, Curr. Opin. Microbiol. 1: 319-329; Carreras and Santi, 1998, Curr. Opin. Biotech. 9: 403-411; and U.S. Pat. Nos. 5,712,146 and 5,672,491, each of which is incorporated herein by reference). This interest has resulted in the cloning, analysis, and manipulation by recombinant DNA technology of genes that encode PKS enzymes. The resulting technology allows one to manipulate a known PKS gene cluster either to produce the polyketide synthesized by that PKS at higher levels than occur in nature or in hosts that otherwise do not produce the polyketide. The technology also allows one to produce molecules that are structurally related to, but distinct from, the polyketides produced from known PKS gene clusters.
There has been a great deal of interest in expressing polyketides produced by Type I and Type II PKS enzymes in host cells that do not normally express such enzymes. For example, the production of the fungal polyketide 6-methylsalicylic acid (6-MSA) in heterologous E. coli, yeast, and plant cells has been reported. See Kealey et al., January 1998, Production of a polyketide natural product in nonpolyketide-producing prokaryotic and eukaryotic host, Proc. Natl. Acad. Sci. USA 95:505-9, U.S. Pat. No. 6,033,883, and PCT Patent Publication Nos. 98/27203 and 99/02669, each of which is incorporated herein by reference. Heterologous production of 6-MSA required or was considerably increased by co-expression of a heterologous acyl carrier protein synthase (ACPS) and that, for E. coli, media supplements were helpful in increasing the level of the malonyl CoA substrate utilized in 6-MSA biosynthesis. See also, PCT Patent Publication No. 97/13845, incorporated herein by reference.
The biosynthesis of other polyketides requires substrates other than or in addition to malonyl CoA. Such substrates include, for example, propionyl CoA, 2-methylmalonyl CoA, 2-hydroxymalonyl CoA, and 2-ethylmalonyl CoA. Of the myriad host cells possible for utilization as polyketide producing hosts, many do not naturally produce such substrates. In addition, to the manipulation of starting substrates and extender molecules there are manipulations of tailoring enzymes in various hosts to achieve diverse polyketide products. Furthermore, the host plays a significant role in polyketide congener production.
Bacteria are classified as strict aerobes (oxygen is necessary for growth), facultative anaerobes (oxygen is not necessary, but results in faster growth), aero tolerant anaerobes (oxygen has no effect on growth), strict anaerobes (oxygen prevents growth), or as microaerophilic (low concentrations of oxygen are required for growth). Oxygen-dependent intracellular processes in aerobic bacteria are mediated by oxidase and oxygenase enzymes. Most myxobacteria and actinomycetes are examples of strict aerobes. Growth of aerobic bacteria under microaerobic (low oxygen) conditions is of interest, as the culture's growth behavior and production of primary and secondary metabolites is often different than that typically observed under a condition of excess oxygenation (Winkelheusen et al., 1996; Schneider et al. 1999; Jensen et al., 2001; Liefke et al., 1990; Kaiser et al., 1994; Dick et al., 1994). References cited by author and year of publication are given a full citation below and each is herein incorporated by reference in its entirety.
Many secondary metabolites produced by members of the Actinomycetales and Myxococcales exhibit potent and varied biological activities. Polyketides are a class of these natural products produced by large multifunctional enzymes called polyketide synthases (PKS), often followed by modification via a subsequent enzymatic pathway (Carreras et al., 2000). The actions of these tailoring pathway enzymes can include glycosylations, hydroxylations, methylations, epoxidations, or a number of other changes to the core structure of the molecule.
Intermediate compounds along these tailoring pathways often possess biological potencies different from the final compound. Examples of this are the antibacterial compounds erythromycins B, C, and D, which can be processed into the more potent erythromycin A congener via an enzymatic pathway which includes the oxygen-dependent EryK hydroxylase ( see FIG. 1). Erythromycin B and its 13-substituted analogs are useful as precursors for the semisynthetic production of prokinetic gastrointestinal agents called motilides. However, erythromycin B is a very poor substrate for the EryK enzyme, and is essentially a shunt end product of this pathway (Lambalot et al., 1995). The ultimate fate of an erythromycin D molecule is then dependent upon a competitive reaction between the EryG methylase and the oxygen dependent EryK hydroxylase. It has been shown previously that the dissolved oxygen concentration during S. erythraea bioconversions of 6-deoxyerythronolide B (6-dEB) analogs is critical in determining if the erythromycin A or B congener is the predominant end product of these cultivations (Carreras et al., 1990).
The epothilones (FIG. 2) have recently generated interest as potential chemotherapeutic successors to the potent anticancer compound paclitaxel (Bollag et al., 1995, Gerth et al., 1996). Both paclitaxel (Taxol®) and the epothilones stabilize microtubules via the same mechanism of action, but the epothilones are effective against paclitaxel-resistant tumors and are more water-soluble (Kowalski et al., 1997, Su et al., 1997). At least 39 different epothilone variants and related compounds have been identified in the fermentation broth of the wild type producing organism, Sorangium cellulosum (Hardt et al.,2001). Epothilone D is reported to possess the highest therapeutic index of the four major epothilone congeners A, B, C, or D (Chou et al., 1998, Chou et al., 2001), but is produced in very low quantity by S. cellulosum (Gerth et al., 2000). The acyl transferase 4 (AT4) domain of the epothilone PKS can incorporate either a malonyl-CoA or a methylmalonyl-CoA extender unit, with this selectivity influencing the final ratio between epothilones A and B, or between epothilones C and D (Gerth et al., 2000). The epothilone D congener is a direct precursor for epoxidation into epothilone B (FIG. 3), catalyzed by the EpoK monooxygenase (Julien et al., 2000, Gerth, et al., 2001).
Erythromycin B and epothilone D are presented as examples of intermediates of secondary metabolite tailoring pathways in which the intermediate is sometimes the desired product. Additionally, there are many other examples of natural products in which a monooxygenase enzyme reaction is necessary for processing of a pathway intermediate into the end product. Examples include the bioconversion of compactin into the cholesterol-lowering drug, pravastatin (Serizawa et al., 1991, Watanabe et al., 1995; the production of the plant hormone, gibberellin (Tudzynski et al., 1998); the production of a fungal mycotoxin, sterigmatocystin (Keller et al., 2000); and the production of doxorubicin, an anti-cancer agent (Lomovskaya et al., 1999, Walczak et al. 1999).
The activity of these oxygen-dependent enzymes can be eliminated by genetic means, but this engineering may prove difficult or impossible to accomplish for various reasons. Directed genetic inactivation of a monooxygenase enzyme requires knowledge of and access to its sequence, as well as the ability to manipulate the DNA of the host organism. The present invention provides an alternate method for producing these intermediates as the major products, without requiring any genetic manipulation of the tailoring enzymes.
Given the potential for making valuable and useful polyketides in large quantities in heterologous host cells, there is a need for host cells capable of making polyketides in the modified distribution of congeners. The present invention helps meet that need by providing recombinant host cells, expression vectors, and methods for making polyketides in diverse host cells.
The following references provide background information on the present invention and are herein incorporated by reference in their entirety.    Winkelhausen, E.; Pittman, P.; Kuzmanova, S.; Jeffries, T. Xylitol formation by Candida boidinii in oxygen limited chemostat culture. Biotechnol. Lett. 1996, 18, 753-758.    Schneider, S.; Wubbolts, M.; Oesterhelt, G.; Sanglard, D.; Witholt, B. Controlled regioselectivity of fatty acid oxidation by whole cells producing cytochrome P450BM-3 monooxygenase under varied dissolved oxygen concentrations. Biotechnol. Bioeng. 1999, 64, 333-341.    Jensen, N.; Melchiorsen, C.; Jokumsen, K.; Villadsen, J. Metabolic behavior of Lactococcus lactis MG1363 in mcroaerobic continuous cultivation at a low dilution rate. Appl. Environ. Microbiol. 2001, 67, 2677-2682.    Liefke, E.; Kaiser, D.; Onken, U. Growth and product formation of actinomycetes cultivated at increased total pressure and oxygen partial pressure. Appl. Environ. Microbiol. 1990, 32, 674-679.    Kaiser, D.; Onken, U.; Sattler, I.; Zeeck, A. Influence of increased dissolved oxygen concentration on the formation of secondary metabolites by manumycin-producing Streptomyces parvulus. Appl. Environ. Microbiol. 1994, 41, 309-312.    Dick, O.; Onken, U.; Sattler, I.; Zeeck, A. Influence of increased dissolved oxygen concentration on productivity and selectivity in cultures of a colabomycin-producing strain of Streptomyces griseoflavus. Appl. Environ. Microbiol. 1994, 41, 373-377.    Carreras, C.; Ashley, G. W. Manipulation of Polyketide Biosynthesis for New Drug Discovery. In New Approaches to Drug Development; Jolles, P., Ed.; Birkhauser-Verlag, Switzerland, 2000; pp 89-108.    Lambalot, R. H.; Cane, D. E.; Aparicio, J. J.; Katz, L. Overproduction and characterization of the erythromycin C-12 hydroxylase, EryK. Biochemistry 1995, 34, 1858-1866.    Carreras, C.; Frykman, S.; Ou, S.; Cadapan, L.; Zavala, S.; Woo, E.; Leaf, T.; Carney, J.; Burlingame, M.; Patel, S.; Ashley, G.; Licari, P. J. Saccharopolyspora erythraea-catalyzed bioconversion of 6-deoxyerythronolide B analogs for production of novel erythromycins. J. Biotechnol. 2002, 92, 217-228.    Bollag, D. M.; McQueney, P. A.; Zhu, J.; Hensens, O.; Koupal, L.; Liesch, J.; Goetz, M.; Lazarides, E.; Woods, C. M. Epothilones, a new class of microtubule-stabilizing agents with a taxol-like mechanism of action. Cancer Res. 1995, 55, 2325-2333.    Gerth, K.; Bedorf, N.; Höfle, G.; Irschik, H.; Reichenbach, H. Epothilons A and B: Antifungal and cytotoxic compounds from Sorangium cellulosum (myxobacteria)—production, physico-chemical, and biological properties. J. Antibiot. 1996, 49, 560-563.    Kowalski, R. J.; Giannakakou, P.; Hamel, E. Activities of the microtubule-stabilizing agents epothilones A and B with purified tubulin and in cells resistant to paclitaxel. J. Biol. Chem. 1997, 272, 2534-41.    Su, D. S.; Meng, D.; Bertinato, P.; Balog, A.; Sorensen, E. J.; Danishefsky, S. J.; Zheng, Y. H.; Chou, T. C.; He, L.; Horwitz, S. B. Structure-activity relationships of the epothilones and the first in vivo comparison with paclitaxel. Angew. Chem. Int. Ed. Engl. 1997, 36, 2093-2096.    Hardt, I.; Steinmetz, H.; Gerth, K.; Sasse, F.; Reichenbach, H.; Höfle, G. New natural epothilones from Sorangium cellulosum, strains So ce90/B2 and So ce90/D13: Isolation, structure elucidation, and SAR studies. J. Nat. Products. 2001, 64, 847-856.    Chou, T. C.; Zhang, X. G.; Balog, A.; Su, D. S.; Meng, D.; Savin, K.; Bertino, J. R.; Danishefsky, S. J. Desoxyepothilone B: An efficacious microtubule-targeted antitumor agent with a promising in vivo profile relative to epothilone B. Proc. Natl. Acad. Sci. USA 1998, 95, 9642-9647.    Chou, T. C.; O'Connor, O. A.; Tong, W. P.; Guan, Y.; Zhang, Z.; Stachel, S. J.; Lee, C.; Danishefsky, S. J. The synthesis, discovery, and development of a highly promising class of microtubule stabilization agents: Curative effects of desoxyepothilones B and F against human tumor xenografts in nude mice. Proc. Natl. Acad. Sci. USA 2001, 98, 8113-8118.    Gerth, K.; Steinmetz, H.; Höfle, G.; Reichenbach, H. Studies on the biosynthesis of epothilones: the biosynthetic origin of the carbon skeleton. J. Antibiot. 2000, 53, 1373-1377.    Julien, B.; Shah, S.; Zierman, R.; Goldman, R.; Katz, L.; Khosla, C. Isolation and characterization of the epothilone biosynthetic gene cluster from Sorangium cellulosum. Gene. 2000, 249, 153-160.    Gerth, K.; Steinmetz, H.; Höfle, G.; Reichenbach, H. Studies on the biosynthesis of epothilones: The PKS and epothilone C/D monooxygenase. J. Antibiot. 2001, 54, 144-148.    Serizawa, N.; Matsuoka, T. A two component-type cytochrome P-450 monooxygenase system in a prokaryote that catalyzes hydroxylation of ML-236B to pravastatin, a tissue-selective inhibitor of 3-hydroy-3-methylglutaryl coenzyme A reductase. Biochem. Biophys. Acta. 1991, 1084, 35-40.    Watanabe, I.; Nara, F.; Serizawa, N. Cloning, characterization and expression of the gene encoding cytochrome P-450sca-2 from Streptomyces carbophilus involved in production of pravastatin, a specific HMG-CoA reductase inhibitor. Gene. 1995, 163, 81-85.    Tudzynski, B.; Holter, K. Gibberellin biosynthetic pathway in Gibberella fujikuroi: evidence of a gene cluster. Fungal Genet. Biol. 1998, 25, 157-170.    Keller, N.; Watanabe, C.; Kelkar, H.; Adams, T.; Townsend, C. Requirement of monooxygenase-mediated steps for sterigmatocystin biosynthesis by Aspergillus nidulans. Appl. Environ. Microbiol. 2000, 66, 359-362.    Lomovskaya, N.; Otten, S.; Doi-Katayama, Y.; Fonstein, L.; Liu, X.; Takatsu, T.; Inventi-Solari, A.; Filippini, S.; Torti, F.; Columbo, A.; Hutchinson, C. R. Doxorubicin overproduction in Streptomyces peucetius: cloning and characterization of the dnrU ketoreductase and dnrV genes and the doxA cytochrome P-450 hydroxylase gene. J. Bacteriol. 1999, 181, 305-318.    Walczak, R.; Dickens, M.; Priestley, N.; Strohl, W. Purification, properties, and characterization of recombinant Streptomyces sp. Strain C5 DoxA, a cytochrome P-450 catalyzing multiple steps in doxorubicin biosynthesis. J. Bacteriol. 1999, 181, 298-304.    Julien, B.; Shah, S. Heterologous expression of the epothilone biosynthetic genes in Myxococcus xanthus. Antimicrob. Agents Chemo. In press.    Pirt, S. J. Oxygen Demand and Supply. In Principles of Microbe and Cell Cultivation. John Wiley and Sons, New York, 1975; pg. 84.    Lau, J.; Frykman, S.; Regentin, R.; Ou, S.; Tsuruta, H.; Licari, P. Optimizing the heterologous production of epothilone D in Myxococcus xanthus. Biotechnol. Bioeng. 2002, 78, 280-288.    Arras, T.; Schirawski, J.; Unden, G. Availability of O2 as a substrate in the cytoplasm of bacteria under aerobic and microaerobic conditions. J. Bacteriol. 1998, 180, 2133-2136.    Tang, L.; Shah, S.; Chung, L.; Carney, J.; Katz, L.; Khosla, C.; Julien, B. Cloning and heterologous expression of the epothilone gene cluster. Science 2000, 287, 640-642.    Frykman, S.; Tsuruta, H.; Lau, J.; Regentin, R; Ou, S; Reeves, C.; Carney, J.; Santi, D.; Licari, P. Modulation of epothilone analog production through media design. J. Ind. Microbiol. Biotechnol. 2002, 28, 17-20.